RNA modification and the regulation of partial EMT in head and neck cancer

头颈癌RNA修饰及部分EMT调控

• 批准号: 10285358
• 负责人: Sidharth Venkata Puram
• 金额: $ 19.69万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10285358
• 关键词: Binding; Biochemical; Biological; Biology; Cancer Etiology; Cell Line; Cells; Clinical; Complex; Critical Pathways; Data; Decision Making; Diagnosis; Disease; Drug Design; Epithelial; Failure; Future; Genes; Genetic Transcription; Goals; Head and Neck Cancer; Head and Neck Squamous Cell Carcinoma; Immunoprecipitation; In Vitro; Investigation; Maps; Mass Spectrum Analysis; Measures; Mesenchymal; Messenger RNA; Methods; Methyltransferase; Modeling; Modification; Neoplasm Metastasis; Nodal; Nuclear; Nuclear Export; Nucleotides; Operative Surgical Procedures; Organoids; Outcome; Pathway interactions; Patient-Focused Outcomes; Patients; Play; Post-Translational Protein Processing; Process; Proteins; RNA; Radiation; Reader; Regulation; Research Proposals; Resistance; Ribonucleosides; Role; Signal Transduction; Smoking Status; Structure; Techniques; Tertiary Protein Structure; Testing; Therapeutic; Transcript; Transcriptional Regulation; Translations; Tumor Cell Invasion; Validation; Writing; alcohol exposure; attributable mortality; base; chemotherapy; crosslink; epithelial to mesenchymal transition; epitranscriptomics; genetic regulatory protein; improved; insight; lymph nodes; mortality; new therapeutic target; novel; patient derived xenograft model; polysome profiling; pre-clinical; prognostic value; programs; single-cell RNA sequencing; small molecule; therapeutic target; tobacco exposure; transcription factor; treatment response; tumor; tumor heterogeneity; tumorigenesis

PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer-related mortality, with the majority of deaths attributable to tumor metastasis and failures in treatment. Because most cases of HNSCC result from tobacco and alcohol exposure, these tumors are highly heterogeneous, greatly complicating diagnosis, treatment, and investigations into the biology of this disease. We recently performed single cell RNA- sequencing (scRNA-seq) in HNSCC and identified a partial epithelial-to-mesenchymal (p-EMT) transcriptional program that is predictive of poor clinical outcomes including nodal metastasis and diminished survival (Puram et al., Cell). Understanding the regulatory factors that control the p-EMT program in HNSCC is of critical importance as targeting multiple genes in a complex pathway such as p-EMT is particularly challenging, yet has the potential to significantly improve HNSCC outcomes and treatment decision-making. RNA modification proteins, which can directly read/write nucleotide marks on RNA, have emerged as one exciting class of such regulatory proteins. Because these proteins modulate multiple RNA transcripts, targeting RNA modification proteins may disrupt expression of multiple disease-related genes and make resistance less likely to emerge. In HNSCC, we have found that METTL3, an RNA methyltransferase which catalyzes the N6-methyladenosine (m6A) modification, is a key regulator of the p-EMT program in HNSCC. In preliminary studies, we have discovered that perturbation of METTL3 in HNSCC disrupts p-EMT signaling and reduces invasion in vitro. However, the precise mechanisms by which METTL3 and other RNA modification proteins exert their function are poorly understood, but may depend on changes in transcript stability and/or translation. Thus, a better understanding of how METTL3 modulates p-EMT in HNSCC is likely to improve rational drug design and future small molecule- and biologically-based screens in search of effective epitranscriptomic therapeutics. We hypothesize that METTL3 antagonizes p-EMT in HNSCC by disrupting the translation of critical p-EMT target genes. To test which domains in METTL3 are essential for its function, we will first perform sophisticated structure-function analyses in HNSCC cell lines and patient-derived xenograft organoid (PDXOs) (Aim 1). We will determine which domains are required for m6A function using state-of-the-art mass spectrometry methods, while also mapping the binding partners of the critical domains. To determine if METTL3 controls the transcription or translation (or both) of p-EMT genes, we will utilize advanced biochemical techniques including meRIP-seq to study the stability of p-EMT RNA transcripts and polysome profiling and PAR-CLIP to investigate the effects of METTL3 on translation of p-EMT genes (Aim 2). These studies will provide indispensable insight into the mechanism by which METTL3 directs HNSCC tumorigenesis, specifically focusing on its regulation of p-EMT signaling, and thereby revealing METTL3 domains that could be targeted by new therapeutics to more effectively and specifically treat HNSCC.

项目摘要 头颈部鳞状细胞癌（HNSCC）是癌症相关死亡的第六大原因， 大多数死亡可归因于肿瘤转移和治疗失败。因为大多数HNSCC病例 由于烟草和酒精暴露，这些肿瘤是高度异质性的，非常复杂， 诊断，治疗和调查这种疾病的生物学。我们最近进行了单细胞RNA- 在HNSCC中进行scRNA-seq测序，并鉴定了部分上皮间质（p-EMT）转录因子。 一个预测包括淋巴结转移和生存率降低在内的不良临床结局的程序（Puram 例如，Cell）。了解控制HNSCC中p-EMT程序的调节因素至关重要 靶向复杂途径如p-EMT中的多个基因的重要性特别具有挑战性，但 显著改善HNSCC结局和治疗决策的潜力。RNA修饰 蛋白质可以直接读/写RNA上的核苷酸标记，已经成为一类令人兴奋的蛋白质， 调节蛋白由于这些蛋白质调节多种RNA转录物，靶向RNA修饰 蛋白质可能会破坏多种疾病相关基因的表达，使耐药性不太可能出现。在 HNSCC，我们发现胃L3，一种催化N6-甲基腺苷 (m6A)修饰，是HNSCC中p-EMT程序的关键调节剂。在初步研究中， 发现HNSCC中胃L3的扰动破坏p-EMT信号传导并降低体外侵袭。 然而，胃L3和其他RNA修饰蛋白发挥其功能的精确机制， 对这些变化的了解很少，但可能取决于转录物稳定性和/或翻译的变化。一个更好的 了解胃L3如何调节HNSCC中的p-EMT可能会改善合理的药物设计， 未来基于小分子和生物学的筛选， 治疗学我们推测，胃L3通过破坏p-EMT的翻译来拮抗HNSCC中的p-EMT。 关键的p-EMT靶基因。为了测试胃L3中的哪些结构域对其功能是必不可少的，我们将首先进行 HNSCC细胞系和患者来源的异种移植类器官（PDXO）的复杂结构-功能分析 (Aim 1）。我们将使用最先进的质谱法确定m6 A功能所需的结构域 方法，同时还映射关键域的结合伴侣。为了确定胃L3是否控制 p-EMT基因的转录或翻译（或两者），我们将利用先进的生化技术，包括 meRIP-seq用于研究p-EMT RNA转录物的稳定性和多核糖体分析，PAR-CLIP用于研究 目的2：研究胃L3对p-EMT基因翻译的影响。这些研究将提供不可或缺的见解 研究胃L3指导HNSCC肿瘤发生的机制，特别关注其对HNSCC肿瘤发生的调控。 p-EMT信号传导，从而揭示了可被新疗法靶向的胃L3结构域， 有效且特异性地治疗HNSCC。